In humans, four important glycoprotein hormones (LH, FSH, TSH and CG) are heterodimers that have identical a subunits and differing β subunits. Three of these hormones are present in virtually all other vertebrate species as well; CG has so far been found only in primates and in the placenta and urine of pregnant mares.
PCT application WO90/09800, published 7 Sep. 1990, and incorporated herein by reference, describes a number of modified forms of these hormones. One important modification is C-terminal extension of the β subunit by the carboxy terminal peptide (CTP) of human chorionic gonadotropin or a variant thereof. Other muteins of these hormones are also described. CTP is the sequence of amino acids extending from any one of positions 112-118 to position 145 of the β subunit of human chorionic gonadotropin. The PCT application describes variants of the CTP extension obtained by conservative amino acid substitutions such that the capacity of the CTP to alter the clearance characteristics of the hormone is not destroyed. In addition, PCT application WO94/24148 published 27 Oct. 1994, incorporated herein by reference, describes modifying these hormones by extension or insertion of the CTP at locations other than the C-terminus and CTP fragments shorter than the sequence extending from positions 112-118 to 145.
The CTP-extended β subunit of FSH is also described in two papers by applicants herein: LaPolt, P. S. et al.; Endocrinology (1992) 131:2514-2520 and Fares, F. A. et al.; Proc Natl Acad Sci USA (1992) 89:4304-4308. Both of these papers are incorporated herein by reference.
The crystal structure of human chorionic gonadotropin has been published in more or less contemporaneous articles; one by Lapthorn, A. J. et al. Nature (1994) 369:455-461 and the other by Wu, H. et al. Structure (1994) 2:545-558. The results of these articles are summarized by Patel, D. J. Nature (1994) 369:438-439.
PCT application WO91/16922 published 14 Nov. 1991 describes a multiplicity of chimeric and otherwise modified forms of the glycoprotein hormones. In general, the disclosure is focused on chimeras of α subunits or β subunits involving portions of various α or β chains respectively. One construct simply listed in this application, and not otherwise described, fuses substantially all of the β chain of human chorionic gonadotropin to the β subunit preprotein, i.e., including the secretory signal sequence for this subunit.
Two additional published PCT applications describe single-chain forms of these glycoprotein hormones wherein the α and β subunits are covalently linked to result in a compound of the general formula:β(linker)nα;orα(linker)nβ
wherein n is 0 or 1 and α and β represent the respective subunits of these hormones: Moyle, W. R., PCT application WO95/22340 published 24 Aug. 1995 and the application of the inventor herein, WO96/05224 published 22 Feb. 1996. The disclosure of these documents is also incorporated herein by reference.
Forms of the above-described single-chain forms of these hormones in which the number of cystine bridges has been depleted are disclosed in U.S. Ser. No. 08/933,693 filed 19 Sep. 1997, and incorporated herein by reference.
The α subunit of a single-chain form of a glycoprotein hormone, CGβ-α, was found to bind noncovalently to an FSHβ subunit as disclosed by the applicants in Society for the Study of Reproduction, Abstract 193, 1996.
Recently, the α subunit of the single-chain glycoprotein hormone, FSHβ-α, was found to form a noncovalent link with a GCβ subunit as disclosed by the applicants in Endocrine Society, Abstract OR28-3, 1998.
It has now been found possible to use these glycoprotein hormones which have enhanced agonist and/or antagonist activity and/or which are multi-functional by either covalently linking an additional β subunit to a single-chain hormone or noncovalently linking an additional β subunit to the tethered α subunit of a single-chain hormone to mimic a natural hormone profile and/or control hormone ratios. These differentially acting glycoprotein hormones and their therapeutic uses for treating disorders such as polycystic ovarian disease, infertility, and ovarian hyperstimulation are disclosed hereinbelow.